Use of a tas2r4 agonist in the preparation of a medicament for the treatment and prevention of diabetic nephropathy

Abstract

The application provides application of a TAS2R4 agonist in preparation of a drug for treating and preventing diabetic nephropathy, and comprises that a bitter taste receptor 4 subtype (TAS2R4) can be used as a drug target point for treating and preventing diabetic nephropathy, and quinine is used as a TAS2R4 agonist. Specifically, mouse diabetic nephropathy induced by a chemical reagent and mouse podocyte cell line MPC cell damage caused by chronic high glucose are taken as research objects, it is found that mouse TAS2R4 agonist quinine has a prevention and treatment effect on diabetic nephropathy, has a protection effect on podocyte loss caused by high glucose, and has an activation effect on mouse kidney TAS2R4 molecular signal, and a possible molecular mechanism is discussed from the aspects of inhibiting NLRP3 inflammasome activation and NF-kappa B signal activation path, and theoretical basis and technical support are provided for prevention and treatment of diabetic nephropathy, other kidney diseases with inactivation of TAS2R4 molecular signal and other diseases by using TAS2R4 agonists including quinine.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to the application of a TAS2R4 agonist in the preparation of drugs for the treatment and prevention of diabetic nephropathy. Background Technology

[0002] Chronic complications of diabetes mellitus (DM) are the leading cause of death and disability in diabetic patients, and diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes and the leading cause of end-stage renal disease (ESRD). Early DN is primarily a glomerular lesion, characterized by glomerular hypertrophy, thickening of the glomerular basement membrane, increased glomerular filtration rate, and abnormal accumulation of extracellular matrix components. Due to its numerous influencing factors and complex pathogenesis, DN has always been a hot topic and a challenging area of ​​research in this field.

[0003] Taste strongly influences food preferences and intake. In vertebrates, the receptors for food or compounds are different taste receptors (TASRs), initially discovered in the taste buds of the oral cavity. Human TASRs include two types: sweet taste receptors and bitter taste receptors (Type 2 taste receptors, TAS2Rs). Bitter compounds activate TAS2Rs. Currently, 25 human TAS2Rs and 35 mouse TAS2Rs (mTAS2Rs) are known. TAS2Rs are also widely distributed in extraoral tissues, such as the respiratory, gastrointestinal, brain, cardiac, urinary, and skin systems. TAS2Rs are a novel class of G protein-coupled receptors (GPCRs), composed of a hydrophobic seven-transmembrane region, a short extracellular N-terminus, an intracellular C-terminus, three extracellular loop structures, and three intracellular loop structures. The TAS2R signaling pathway in the taste system is highly conserved. Upon activation by ligands, TAS2Rs dissociate from the G protein, leading to the dissociation of the G protein Gαβγ complex and the formation of two pathways for their function: the Gαgust-PDE-cAMP-PKA pathway and the Gβγ-PLCβ-IP3-Ca pathway. 2+ -TRPM5 and Gβγ-PLCβ-DAG-PKC pathways can ultimately lead to the release of neurotransmitters, hormones, etc.

[0004] In the current technology, the research on bitter taste receptors TAS2Rs in extraoral tissues is still limited to the role of TAS2Rs as immune sentinels in the body, which have the body's defense function. In particular, TAS2Rs can sense bitter components in food and microbial metabolites (harmful or potentially toxic) that are widely present in nature, such as the respiratory tract, digestive tract, urinary and reproductive tract, and blood-brain barrier, which are distributed in organs that are in close contact with the outside world or have barrier functions.

[0005] The kidneys play a vital role in the body's elimination of harmful substances and defense functions, and chronic inflammation has been widely recognized as one of the important mechanisms leading to chronic kidney disease.

[0006] According to Rajkumar P, et al., PloS One A study published in 2014, 9: e111053, reported the expression of some TAS2R subtypes in mouse kidneys. The vast majority of TAS2R gene transcripts were detectable in primary mouse renal tubular and collecting duct cell lines, as well as in the kidneys of mice 40 days after birth (Jie Liang, et al.). Mol Cell Biochem (2017 Apr;428(1-2):193-202.). However, little is known about the role of TAS2Rs in kidney disease and the biological functions exhibited by their activation. It has been reported that carriers of the TAS2R43 allele are protected from Balkan endemic kidney disease. However, it remains unknown whether kidney-expressed TAS2Rs and their mediated molecular signaling are involved in the development and progression of diabetic nephropathy. Summary of the Invention

[0007] To address the technical problems existing in the prior art, this invention discloses the application of a TAS2R4 agonist in the preparation of drugs for the treatment and prevention of diabetic nephropathy. The renal TAS2R4 receptor can be used as a new target for drugs to treat and prevent chronic kidney diseases such as diabetic nephropathy. TAS2R4 agonists, represented by quinine, are applied to drugs for the prevention and treatment of chronic kidney diseases such as diabetic nephropathy.

[0008] To achieve the above objectives, this invention provides the application of a TAS2R4 agonist in the preparation of drugs for the treatment and prevention of diabetic nephropathy. Specifically, the bitter taste receptor subtype 4 (TAS2R4) can serve as a drug target for the treatment and prevention of diabetic nephropathy. TAS2R4 agonists exhibit a wide range and diversity in chemical structure, including but not limited to alkaloids, terpenes, saponins, conjugated quinones, ketones, polypeptides, amino acids, and sterols.

[0009] Preferably, quinine is used as a TAS2R4 agonist. Specifically, the study focuses on mouse DN induced by chemical reagents and mouse podocyte MPC cell damage caused by chronic hyperglycemia. The TAS2R4 agonist quinine has a preventive and protective effect against DN and a protective effect against podocyte loss caused by hyperglycemia. It also activates TAS2R4 molecular signaling, thereby inhibiting NLRP3 inflammasome activation and NF-κB signaling activation.

[0010] Preferably, the TAS2R4 agonist can activate the renal TAS2R4 receptor signaling.

[0011] Preferably, the TAS2R4 agonist can produce an anti-inflammatory effect by activating the TAS2R4 molecular signaling of glomerular podocytes, thereby reducing damage to glomerular podocytes.

[0012] Preferably, the TAS2R4 agonist can improve fasting blood glucose, body weight, and renal function in diabetic mice, and inhibit the renal NLRP3 inflammasome and NF-κB. κ Activation of B.

[0013] Preferably, the TAS2R4 agonist can restore the decreased cell viability of mouse podocytes caused by high glucose, reverse the decrease in the levels of podocyte marker proteins nephrin and ZO-1, increase the protein expression of TAS2R4 and its downstream signaling molecule phospholipase Cβ2 (PLCβ2), and increase intracellular calcium ion levels.

[0014] Preferably, the TAS2R4 agonist is derived from natural products, chemically synthesized compounds, microbial metabolites, etc.

[0015] This invention is based on the therapeutic and preventive effects of TAS2R4 agonists on diabetic nephropathy. Specifically, activation of TAS2R4 molecular signaling in glomerular podocytes produces an anti-inflammatory effect, thereby alleviating glomerular lesions in diabetic nephropathy. Animal experiments revealed that quinine, a typical TAS2R4 agonist, improved fasting blood glucose, body weight, and renal function in diabetic mice, reduced glomerular podocyte damage, and inhibited the activation of the renal NLRP3 inflammasome and NF-κB. Quinine also activated the renal TAS2R4 receptor signaling. Cellular experiments showed that the gene encoding the mouse TAS2R4 protein... Tas2r108The TAS2R4 receptor exhibits high gene expression abundance in mouse podocytes, prompting us to investigate its biological function in podocyte TAS2R4 receptor activation under chronic hyperglycemia. On one hand, the TAS2R4 agonist quinine restored cell viability in mouse podocytes induced by hyperglycemia, reversed the decrease in podocyte marker proteins nephrin and ZO-1, and increased TAS2R4 protein expression, as well as the protein expression of its downstream signaling molecule phospholipase Cβ2 (PLCβ2) and intracellular calcium ion levels. On the other hand, TAS2R4 receptor antagonism (treatment with two TAS2R4 receptor antagonists, abscisic acid (ABA) and gamma-aminobutyric acid (GABA)) attenuated the effects of quinine in increasing PLCβ2 protein expression and reducing NF-κB p65 phosphorylation. Simultaneously, the TAS2R4 antagonist ABA, the Gβγ inhibitor Gallein, and the PLCβ2 inhibitor U73122 reversed the effects of quinine in reducing NLRP3 and Cleaved caspase 1 protein expression. In summary, the quinine provided by this invention, as a TAS2R4 agonist, can produce an anti-inflammatory effect by activating the TAS2R4 molecular signaling of glomerular podocytes, thereby improving podocyte damage caused by chronic hyperglycemia and having a preventive and therapeutic effect on diabetic nephropathy and other kidney diseases with inactivated TAS2R4 molecular signaling.

[0016] In summary, this invention uses quinine as an agonist to activate the bitter taste receptor subtype 4 (TAS2R4) in the kidney, thereby playing a key role in the treatment and prevention of diabetic nephropathy. On the other hand, quinine is prepared as an agonist into capsules, sugar-coated tablets, etc., for practical application in therapeutic and preventive drugs.

[0017] Technical effects of the present invention:

[0018] 1. Using the technical solution of this invention, this study investigated the preventive and therapeutic effects of the TAS2R4 agonist quinine on diabetic nephropathy and podocyte loss, as well as its protective effect against TAS2R4 molecular signaling, using chemically induced diabetic nephropathy and chronic hyperglycemia-induced damage to mouse podocyte MPC cells as research subjects. The study also explored possible molecular mechanisms by inhibiting NLRP3 inflammasome activation and NF-κB signaling activation, providing a theoretical basis and technical support for the treatment and prevention of diabetic nephropathy and other kidney diseases with TAS2R4 molecular signaling inactivation.

[0019] 2. In this invention, quinine is used as a TAS2R4 activator. After five weeks of administration, it can improve fasting blood glucose levels and body weight in diabetic mice. After nine weeks of administration, it can significantly improve renal function in diabetic mice, such as reducing plasma creatinine and urea nitrogen levels and urinary protein excretion.

[0020] 3. This invention uses quinine as a TAS2R4 activator. After nine weeks of administration, it not only alleviates glomerular podocyte damage in diabetic mice by increasing the expression of Nephrin, Podocin, and ZO-1 proteins, but also enhances TAS2R4 molecular signaling in the kidneys of diabetic mice by increasing the expression of TAS2R4 and PLCβ2 proteins. At the same time, it also inhibits the activation of NLRP3 inflammasome and NF-κB in the kidneys of diabetic mice by reducing the expression of NLRP3, Cleaved caspase 1, and IL-1β proteins, as well as the expression of NF-κB p65 protein in the cytoplasm.

[0021] 4. This invention uses quinine as a TAS2R4 activator, which can not only increase cell viability, Nephrin and ZO-1 protein expression in mouse podocyte cell line MPC after 48 h of chronic high glucose culture, but also increase TAS2R4 and PLCβ2 protein expression and intracellular calcium ion concentration.

[0022] 5. In this invention, quinine is used as a TAS2R4 activator. When mouse podocyte cell line MPC is co-treated with the TAS2R4 antagonists γ-aminobutyric acid (GABA) and abscisic acid (ABA), the effects of quinine in increasing PLCβ2 protein expression and decreasing p-NF-κB p65 levels in the cytoplasm are significantly reversed. Furthermore, when mouse podocyte cell line MPC is co-treated with the TAS2R4 antagonist abscisic acid, the Gβγ inhibitor Gallein, and the PLCβ2 inhibitor U73122, the effects of quinine in decreasing NLRP3 and Cleaved caspase 1 protein expression are significantly eliminated. Attached Figure Description

[0023] Figure 1 This invention relates to the effect of the TAS2R4 agonist quinine on the expression of ZO-1 protein in the kidneys of DM mice in Example 1 of this invention.

[0024] Figure 2 This invention relates to the effect of the TAS2R4 agonist quinine on the expression of PLCβ2 protein in the kidneys of DM mice in Example 1 of this invention.

[0025] Figure 3 This invention relates to the effect of the TAS2R4 agonist quinine on the expression of Cleaved caspase 1 protein in the kidneys of DM mice in Example 2 of this invention.

[0026] Figure 4 This invention relates to the effect of the TAS2R4 agonist quinine on the expression of NF-κB p65 protein in the kidneys of DM mice in Example 2 of this invention.

[0027] Figure 5This invention relates to the effect of the TAS2R4 agonist quinine on the expression of PLCβ2 protein in the mouse podocyte cell line MPC under high glucose conditions in Example 3 of this invention.

[0028] Figure 6 This invention relates to the effects of TAS2R4 antagonists γ-aminobutyric acid (GABA) and abscisic acid (ABA) on the increase in PLCβ2 protein expression in mouse podocyte cell line MPC under high glucose conditions caused by the TAS2R4 agonist quinine.

[0029] Figure 7 This invention relates to the effects of TAS2R4 antagonists γ-aminobutyric acid (GABA) and abscisic acid (ABA) on the reduction of p-NF-κB p65 protein expression in mouse podocyte cell line MPC under high glucose conditions by the TAS2R4 agonist quinine.

[0030] Figure 8 This invention relates to Example 4, which describes the effects of the TAS2R4 antagonist abscisic acid (ABA), the Gβγ inhibitor Gallein, and the PLCβ2 inhibitor U73122 on the reduction of NLRP3 protein expression in the mouse podocyte cell line MPC under high glucose conditions caused by the TAS2R4 agonist quinine.

[0031] Figure 9 This invention relates to the effects of the TAS2R4 antagonist abscisic acid (ABA) and the Gβγ inhibitor Gallein in Example 4 of this invention on the reduction of activated caspase 1 protein expression in the mouse podocyte cell line MPC under high glucose conditions by the TAS2R4 agonist quinine. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0033] The technical solution of the present invention will be further described in detail below through specific embodiments. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0034] Example 1

[0035] This embodiment illustrates the effects of quinine, a TAS2R4 agonist provided in this invention, on basic indicators of diabetes and renal function in STZ-induced diabetic DN mice.

[0036] 1. Materials and Methods

[0037] 1.1 Animals

[0038] Male C57BL / 6J mice (8-9 weeks old) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0039] 1.2 Medicines and Reagents

[0040] 1.2.1 Drugs: Quinine (130-95-0, purity>99%) was purchased from Beijing Huawi Ruike Chemical Co., Ltd.; Streptozotocin (STZ, S817944, purity>99%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0041] 1.2.2 Main Reagents: Creatinine (Cr) assay kit (C011), blood urea nitrogen (BUN) test kit (C013), and urine protein quantification kit (C035-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute. Nephrin antibody (38552) was purchased from SAB Biotechnology, USA; Podocin antibody (20384-1-AP) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; ZO-1 antibody (21773-1-AP) was purchased from Proteintech; β-actin antibody (ET1701-80) was purchased from Hangzhou Huaan Biotechnology Co., Ltd.; and DyLight594 donkey anti-rabbit IgG antibody (E032421-01) was purchased from EARTHOX, USA. SDS (S8010) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; SDS-PAGE protein loading buffer 5× (P0015), SDS-PAGE gel preparation kit (P0012A), and PMSF (ST506) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; color pre-stained protein marker (26616) was purchased from Thermo Scientific, USA; and NC membrane was purchased from Millipore, USA.

[0042] 1.3 Methods

[0043] 1.3.1 Preparation of STZ-induced diabetic mouse model: First, mice were ear-tagged and weighed the day before modeling, and the weight was recorded. Second, mice were fasted overnight but allowed water for more than 12 hours. A fresh streptozotocin (STZ) solution was prepared according to the required amount (protected from light), and STZ was administered intraperitoneally at a dose of 50 mg / kg for 5 consecutive days to induce a diabetic mouse model. Then, after seven days of feeding, mice were fasted for approximately 8 hours but allowed water. A suitable amount of blood was collected from the tail vein and dripped onto a standardized blood glucose test strip. The fasting blood glucose (FBG) level of the mice was measured using a matched blood glucose meter. Finally, mice with FBG values ​​>11.1 mmol / L were considered successfully modeled diabetic mice and divided into groups.

[0044] 1.3.2 Grouping and Administration: Successfully modeled diabetic mice were randomly divided into two groups: a diabetes model group (DM) and a TAS2R4 agonist quinine group (DM + Quinine). A normal control group was also established, with 10 mice in each group. The administration groups were given quinine (prepared as a suspension in 0.5% CMC-Na) at a dose of 80 mg / kg by gavage (10 mL / kg volume). The model group and the normal control group were given the same volume of 0.5% CMC-Na once daily for 9 consecutive weeks. Body weight was monitored weekly, and the dosage was adjusted accordingly. Fasting blood glucose was monitored once during the administration period and at the time of animal treatment.

[0045] 1.3.3 Urine Protein Assay: Approximately 9 weeks after drug administration, urine was collected from mice using the bladder compression method, and the urine volume was accurately recorded. 0.2 ml of urine was placed in an EP tube and centrifuged at 3000 g for 5 min at 4°C. The supernatant was collected. The supernatant was pre-frozen at -20°C for several hours before being transferred to a -80°C freezer for later use. The urine protein concentration was determined using a urine protein quantification kit and the Coomassie Brilliant Blue method (CBB method), strictly following the instructions.

[0046] 1.3.4 Determination of Plasma Biochemical Indicators: After fasting for 8 hours with free access to water, the mice were enucleated, and blood was collected from the orbital vein. The blood was quickly placed in a 1.5 mL EP tube containing heparin anticoagulant, centrifuged for 5 min at 4℃ and 3000 g to separate the plasma. The supernatant was stored at -80℃ for later use. Cr and BUN levels were determined spectrophotometrically using the corresponding assay kits, strictly following the instructions.

[0047] 1.3.5 Western blotting for protein expression determination: Absorbed and preserved renal cortex samples were separated by electrophoresis, transferred to a nitrocellulose membrane, washed, blocked, reacted with primary and secondary antibodies, developed, and scanned or photographed. Changes in protein expression levels were analyzed using ImageJ software. β-actin was used as an internal control. The proteins measured in this section included Nephrin and Podocin.

[0048] 1.3.6 Immunohistochemical-fluorescence assay for protein expression: The protein measured in this section is ZO-1. The specific steps are as follows:

[0049] 1) Tissue section: 4 μm;

[0050] 2) Dewaxing and hydration of tissue sections: xylene I 15 min, xylene II 15 min, 100% ethanol 5 min, 95% ethanol 5 min, 80% ethanol 5 min;

[0051] 3) Soak in distilled water for 15 min, wash with PBS 3 times for 5 min each time, and wipe off excess liquid;

[0052] 4) Microwave restoration: Place the slide in a pH 6.0 citric acid buffer solution, close the lid, microwave on high for 5 minutes, then on low for 15 minutes. Allow to cool naturally to room temperature. Wash the slide with PBS for 5 minutes each time (3 times), shake off excess liquid, and wipe dry.

[0053] 5) Blocking: Prepare blocking solution by mixing fetal bovine serum and PBS at a ratio of 1:200. Add 40 μL to each tissue to cover the tissue surface and block at room temperature for 40 min.

[0054] 6) Shake off excess liquid from the surface of the tissue section and wipe off the surrounding liquid. Do not wash. Add ZO-1 (1:200) primary antibody dilution solution to the surface of the section and incubate overnight at 4°C in a humidified chamber.

[0055] 7) Remove the humidified box at 4 ℃ and incubate at 37 ℃ for 30 min;

[0056] 8) Washing: Wash with PBS, 5 min × 3 times;

[0057] 9) Wipe the liquid around the tissue dry, add 40 μl of fluorescently labeled secondary antibody (1:200) to each tissue section under light-protected conditions, cover the tissue surface, and incubate at 37 ℃ in the dark for 1 h.

[0058] 10) Discard the liquid, wash the slide with PBS for 5 min × 3 times, add DAPI staining solution, stain for 3 min, wash with PBS for 5 min × 3 times (operate in the dark).

[0059] 11) Add anti-fluorescence quenching mounting solution to the tissue, cover with a coverslip to avoid air bubbles, and fix the four corners with colorless nail polish (operate in the dark).

[0060] 12) Photographs were taken under a fluorescence microscope. The final results showed that the ZO-1 protein exhibited red fluorescence, while the cell nucleus showed blue fluorescence.

[0061] 2 Results

[0062] 2.1 Effects of the TAS2R4 agonist quinine on blood glucose and body weight in diabetic mice

[0063] Five weeks after quinine administration, compared with untreated diabetic diabetic mice, quinine administration significantly reduced FBG levels in diabetic mice (P<0.01, Table 1). Nine weeks after quinine administration, compared with untreated diabetic mice, quinine administration further significantly reduced FBG levels in diabetic mice (P<0.01, Table 1). Throughout the experiment, compared with untreated diabetic mice, quinine administration significantly increased the body weight of diabetic mice (P<0.05, Table 2), and this significant increase continued three weeks after administration (P<0.01, Table 2). These results indicate that TAS2R4 agonists can improve the basic symptoms of diabetic animals.

[0064] Table 1. Effects of the TAS2R4 agonist quinine on fasting blood glucose levels in DM mice.

[0065]

[0066] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group.

[0067] Table 2 Effects of the TAS2R4 agonist quinine on body weight in DM mice

[0068]

[0069] Note: Mean±SD, * P<0.05, compared with the Normal group; # P<0.05, ## P<0.01, compared with the DM group.

[0070] 2.2 Effects of the TAS2R4 agonist quinine on renal function in diabetic mice

[0071] Plasma urea nitrogen levels were significantly increased in DM mice compared to normal mice (P<0.01). The TAS2R4 agonist quinine significantly reduced plasma urea nitrogen levels in DM mice (all P<0.01). Results are shown in Table 3. Plasma creatinine levels were significantly increased in DM mice compared to normal mice (P<0.01). The TAS2R4 agonist quinine significantly reduced plasma creatinine levels in DM mice (P<0.01). Results are shown in Table 4. Urinary protein excretion was significantly increased in DM mice compared to normal mice (P<0.01). The TAS2R4 agonist quinine significantly reduced urinary protein excretion in DM mice (P<0.01). Results are shown in Table 5.

[0072] Table 3 Effects of the TAS2R4 agonist quinine on plasma urea nitrogen levels in DM mice

[0073]

[0074] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group.

[0075] Table 4. Effects of the TAS2R4 agonist quinine on plasma creatinine levels in DM mice.

[0076]

[0077] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group.

[0078] Table 5 Effects of the TAS2R4 agonist quinine on urinary protein levels in DM mice

[0079]

[0080] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group.

[0081] 2.3 Effects of the TAS2R4 agonist quinine on glomerular podocyte function in diabetic mice

[0082] The expression of podocyte marker proteins Nephrin and Podocin in the renal cortex of diabetic mice was significantly decreased compared with that in normal mice (both P < 0.01), as shown in Tables 6 and 7. The TAS2R4 agonist quinine significantly increased the expression of Nephrin and Podocin proteins in the renal cortex of diabetic mice (both P < 0.01), as shown in Tables 6 and 7. Furthermore, the expression of the podocyte tight junction protein ZO-1 in the renal cortex of diabetic mice was significantly decreased, while treatment with the TAS2R4 agonist quinine significantly increased the expression of ZO-1 protein in the renal cortex of diabetic mice, as shown in Tables 6 and 7. Figure 1 .

[0083] Table 6. Effects of the TAS2R4 agonist quinine on Nephrin protein expression in the renal cortex of DM mice.

[0084]

[0085] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group.

[0086] Table 7 Effects of the TAS2R4 agonist quinine on podocin protein expression in the renal cortex of DM mice

[0087]

[0088] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group. Example

[0089] This embodiment provides the effects of the TAS2R4 agonist quinine on TAS2R4 signaling and inflammatory pathways in the renal cortex of STZ-induced DN mice, specifically including:

[0090] 1. Materials and Methods

[0091] 1.1 Animals

[0092] Same as Example 1.

[0093] 1.2 Medicines and Reagents

[0094] 1.2.1 Medicine: Same as in Example 1.

[0095] 1.2.2 Main reagents: TAS2R4 antibody (DF10272) and NLRP3 antibody (DF7438) were purchased from Affinity Pharmaceuticals, USA; PLCβ2 antibody (A8141) was purchased from Abclonal Antibody Company; NF-κB p65 antibody (8242S) was purchased from CellSignaling Technology, USA; Cleaved caspase 1 (D210) antibody (40499) and IL-β antibody (41059) were purchased from Signalway Antibody, USA. Other Western blot reagents and immunohistochemical fluorescence reagents were the same as in Example 1.

[0096] 2 Results

[0097] 2.1 Effects of the TAS2R4 agonist quinine on TAS2R4 signaling in the renal cortex of DM mice

[0098] The expression of TAS2R4 protein in the renal cortex of DM mice was significantly reduced, which was statistically significant compared with that in the renal cortex of normal mice (P<0.01). The results are shown in Table 8.

[0099] Table 8 Effects of the TAS2R4 agonist quinine on TAS2R4 protein expression in the renal cortex of DM mice

[0100]

[0101] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group.

[0102] The TAS2R4 agonist quinine significantly increased TAS2R4 protein expression in the renal cortex of diabetic mice (P<0.01), as shown in Table 8. Simultaneously, PLCβ2 protein expression was significantly decreased in the renal cortex of diabetic mice, while the TAS2R4 agonist quinine significantly increased PLCβ2 protein expression in the renal cortex of diabetic mice, as shown in Table 8. Figure 2 .

[0103] 2.2 Effects of the TAS2R4 agonist quinine on the expression of NLRP3 inflammasome and NF-κB p65 protein in the renal cortex of DM mice

[0104] The expression of NLRP3 and IL-1β proteins in the renal cortex of diabetic mice was significantly increased compared with that in normal mice (both P < 0.01), as shown in Tables 9 and 10. Conversely, the TAS2R4 agonist quinine significantly decreased the expression of NLRP3 and IL-1β proteins in the renal cortex of diabetic mice (both P < 0.01), as shown in Tables 9 and 10. Simultaneously, the expression of Cleaved caspase 1 protein in the renal cortex of diabetic mice was significantly increased, while the TAS2R4 agonist quinine significantly decreased the expression of Cleaved caspase 1 protein in the renal cortex of diabetic mice, as shown in Tables 9 and 10. Figure 3 Furthermore, NF-κB p65 protein expression was significantly increased in the renal cortex of DM mice, while the TAS2R4 agonist quinine significantly decreased NF-κB p65 protein expression in the renal cortex of DM mice. (See results below.) Figure 4 .

[0105] Table 9 Effects of the TAS2R4 agonist quinine on NLRP3 protein expression in the renal cortex of DM mice

[0106]

[0107] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group.

[0108] Table 10 Effects of the TAS2R4 agonist quinine on IL-1β protein expression in the renal cortex of DM mice

[0109]

[0110] Note: Mean±SD, ** P<0.01, compared with the Normal group; ## P<0.01, compared with the DM group. Example

[0111] This embodiment provides the effects of the TAS2R4 agonist quinine on the function and TAS2R4 signaling of mouse podocyte cell line MPC cultured in high glucose, specifically including:

[0112] 1. Materials and Methods

[0113] 1.1 Cell lines

[0114] The mouse glomerular podocyte cell line MPC cells (BNCC337685) were purchased from BeiNa Biotechnology Co., Ltd.

[0115] 1.2 Medicines and Reagents

[0116] 1.2.1 Drugs: Quinine (130-95-0, purity>99%) was purchased from Beijing Huawi Ruike Chemical Co., Ltd.

[0117] 1.2.2 Main Reagents: DMSO (D5879) was purchased from Sigma-Aldrich (USA); CCK-8 kit was purchased from Shanghai Dongren Chemical Technology Co., Ltd.; Fura-2 calcium ion fluorescent probe was purchased from Jiangsu Beyotime Biotechnology Research Institute; BCA protein assay kit (23225) was purchased from Thermo-Scientific (Rockford, IL, USA); rabbit TAS2R4 antibody (DF10272) was purchased from Affinity Antibody Company; rabbit PLCβ2 antibody (A8141) was purchased from ABclonal Antibody Company; rabbit β-actin (AP0060) antibody was purchased from Bioworld Technology Antibody Company; goat anti-rabbit Dylight 594 Affinipure IgG (H+L) secondary antibody (V926-32211) was purchased from Li-Cor, Inc. (Lincoln, NE); mTas2r108, ​​-113, -136, Primers -105, -106, -123, and Actb were synthesized by Shanghai Sangon Biotech Co., Ltd.; RIPA lysis buffer (strong) (P0013B) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; RPMI-1640.0 Medium and fetal bovine serum were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; Trizol reagent (15596-026) was purchased from Invitrogen, USA; SYBR Premix Ex Taq II was purchased from TaKaRa, Japan; Triton X-100 (9005-64-5) and Tween20 (9005-64-5) were purchased from Xuzhou Microcom Biotechnology Co., Ltd. Other reagents used in routine experiments were the same as in Example 1.

[0118] 1.3 Methods

[0119] 1.3.1 Grouping and Drug Administration: MPC cells are adherent cells and were routinely cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin in a 5% CO2 incubator at 37 ℃. When growth was good, the medium was replaced with serum-free medium to synchronize the cell cycle for 12 h. Cells were then divided into a normal glucose group (NG, 11.1 mM), a high glucose group (HG, 40 mM), and a TAS2R4 agonist quinine (Qui) treatment group. Relevant parameters were measured after 48 h of culture.

[0120] 1.3.2 mRNA Level Measurement: mRNA levels were measured using RT-qPCR. Total RNA was extracted using Trizol, and the concentration was determined according to the reagent instructions. Purity was assessed; samples with an OD260 / 280 ratio between 1.8 and 2.0 were considered acceptable. 0.5 μg of total RNA from each sample was used for reverse transcription into cDNA, following the instructions for the cDNA reverse transcription synthesis kit. The synthesized cDNA was amplified using a Roche 480 real-time PCR instrument. After amplification, melting curves were plotted to determine the purity of the amplified fragment. A lower Cp value indicates a higher content of the target gene. The 2-ΔCp value (ΔCp = ΔCpβ-actin - ΔCptarget) was used as the relative value of gene expression in that sample.

[0121] 1.3.3 Cell viability assay: Cell viability was determined using the CCK-8 assay. Following the cell culture procedure in 1.3.1, two concentrations of 50 and 100 µmol / L were set. Cells were collected after 48 h of culture and assayed according to the kit instructions.

[0122] 1.3.4 Calcium Ion Concentration Measurement: Intracellular calcium ion concentration was measured using a fluorescent probe method. Following the cell grouping and culture procedures in 1.3.1, cells were washed three times with PBS, incubated with Fura-2 calcium ion fluorescent probe working solution in the dark for 45 min, and then washed three times with PBS. A fluorescence microplate reader was used to detect the fluorescence intensity at excitation wavelengths of 340 nm and 380 nm, and the emission wavelength of 510 nm. Calcium ion concentration was reflected by the ratio of fluorescence intensity at 340 nm to 380 nm; a higher ratio indicated a higher calcium ion concentration.

[0123] 1.3.5 Protein Expression Assay (Western blot): Protein concentration in cell homogenate supernatant was determined using the BCA method, and the protein solution was diluted with SDS loading buffer. Protein expression was determined using a standard Western blot method, which involved sequential electrophoresis, transfer, blocking, primary antibody incubation, secondary antibody incubation, development, and scanning. The relative grayscale values ​​of the target protein and internal control protein were used as indicators to analyze changes in the target protein expression level.

[0124] 1.3.6 Protein expression assay (Immunofluorescence method): A sterile round coverslip was placed in a 12-well plate, and cells were seeded in the well plate. After the cells grew stably on the coverslip, they were given serum-free medium for cell cycle synchronization treatment for 12 h. When administering drugs, the complete medium was replaced and drugs were administered separately. The medium was discarded after 24 h, and subsequent experiments were carried out.

[0125] 1) Wash the cell smears with PBS, 5 min / time × 3 times;

[0126] 2) Add 4% paraformaldehyde or methanol to cover the slide and fix at -20 °C for 15 min;

[0127] 3) Wash with PBS, 5 min / time × 3 times;

[0128] 4) Add 0.1% Triton X-100 and perforate for 10 min;

[0129] 5) Wash with PBS, 5 min / time × 3 times;

[0130] 6) Add 1% BSA (prepared with PBS) and block for 30 min;

[0131] 7) Dilute the primary antibody according to the instructions, cover the entire slide, and incubate overnight at 4 °C in a humidified chamber.

[0132] 8) Wash with PBS, 5 min / time × 3 times;

[0133] 9) Prepare the Dylight 594 (anti-rabbit) labeled secondary antibody according to the instructions, cover the entire slide, and incubate at 37 °C for 1 h;

[0134] 10) Wash with PBS, 5 min / time × 3 times;

[0135] 11) DAPI staining for 2 minutes;

[0136] 12) Wash with PBS, 5 min / time × 3 times;

[0137] 13) Blot the remaining PBS with filter paper, add a drop of anti-fluorescence quencher, and transfer the slide onto a glass slide with the front side facing down.

[0138] 14) Take photos using a fluorescence microscope to observe the cellular localization of the target protein. The final result shows the target protein as red fluorescence and the cell nucleus as blue fluorescence.

[0139] 2 Results

[0140] 2.1 Comparison of mouse Tas2r gene expression abundance in MPC cells

[0141] After culturing MPC cells for 48 h, the expression of several mouse bitter taste receptor genes was measured using real-time quantitative PCR, and it was found that... Tas2r108 The highest expression level, and Tas2r108 , Tas2r113 , Tas2r136 , Tas2r105 , Tas2r106 , Tas2r123 The mRNA levels decreased sequentially (see Table 11). Since mouse Tas2r108 is 100% orthologous to human TAS2R4, its encoded receptor protein mTAS2R4 largely shares similar biological functions with the TAS2R4 receptor. Therefore, MPC cells can be used for functional studies of human TAS2R4 agonists.

[0142] Table 11 MPC cells of mice Tas2r Gene expression abundance

[0143]

[0144] Note: RT-qPCR method was used to measure mice. Tas2r Horizontal. Mean ± SD, rounded to three significant figures.

[0145] 2.2 Effects of quinine on the biological functions of MPC cells cultured in high glucose

[0146] Compared with the NG group, the MPC cell viability in the HG group was significantly reduced, decreasing by nearly 30% (P<0.01, Table 12). Treatment with both concentrations of quinine significantly increased the MPC cell viability in the HG group (both P<0.01, Table 12), but the effect was stronger in the 50 µmol / L group than in the 100 µmol / L group. Therefore, subsequent experiments used 50 µmol / L quinine.

[0147] Compared with the NG group, the expression of podocyte marker proteins Nephrin (P<0.01, Table 13) and tight junction protein ZO-1 (P<0.05, Table 14) in MPC cells of the HG group was significantly reduced. However, after quinine treatment, the expression of both Nephrin and ZO-1 in MPC cells of the HG group was significantly increased (both P<0.01, Tables 13, 14). These results indicate that TAS2R4 agonists can ameliorate podocyte injury induced by chronic hyperglycemia.

[0148] Table 12 Effects of the TAS2R4 agonist quinine on the viability of mouse podocyte MPC cells under high glucose conditions

[0149]

[0150] Note: Mean±SD, ** P<0.01, compared with NG group; ## P<0.01, compared with HG group.

[0151] Table 13 Effects of the TAS2R4 agonist quinine on Nephrin protein expression in the mouse podocyte cell line MPC under high glucose conditions.

[0152]

[0153] Note: Mean±SD, ** P<0.01, compared with NG group; ## P<0.01, compared with HG group.

[0154] Table 14 Effects of the TAS2R4 agonist quinine on ZO-1 protein expression in mouse podocyte cell line MPC under high glucose conditions

[0155]

[0156] Note: Mean±SD, * P<0.05, compared with NG group; ## P<0.01, compared with HG group.

[0157] 2.3 Effect of quinine on TAS2R4 signaling in MPC cells cultured in high glucose

[0158] Compared with the NG group, TAS2R4 protein expression in MPC cells of the HG group was significantly decreased by 31% (P<0.01, Table 15), but intracellular calcium ion levels did not change significantly (Table 16). After quinine treatment, TAS2R4 protein expression in MPC cells of the HG group significantly increased, almost returning to the level of the NG group (P<0.01, Table 15), and intracellular calcium ion levels in MPC cells of the HG group were significantly increased (P<0.01, Table 16). Meanwhile, PLCβ2 protein expression in MPC cells of the HG group was significantly decreased, while co-treatment with quinine significantly increased PLCβ2 protein expression in MPC cells. (See Table 16 for details). Figure 5 These results indicate that TAS2R4 agonists can activate TAS2R4 signaling in podocytes under chronic hyperglycemic conditions.

[0159] Table 15 Effects of the TAS2R4 agonist quinine on TAS2R4 protein expression in mouse podocyte cell line MPC under high glucose conditions

[0160]

[0161] Note: Mean±SD, ** P<0.01, compared with NG group; ## P<0.01, compared with HG group.

[0162] Table 16 Effects of the TAS2R4 agonist quinine on intracellular calcium ion concentration in mouse podocyte MPC cells under high glucose conditions

[0163]

[0164] Note: Mean±SD, ## P<0.01, compared with HG group.

[0165] Example 4

[0166] This embodiment provides a study on the effect and mechanism of TAS2R4 signal inhibition on quinine's improvement of MPC cell damage induced by high glucose.

[0167] 1. Materials and Methods

[0168] 1.1 The cell line is the same as in Example 3.

[0169] 1.2 Medicines and Reagents

[0170] 1.2.1 Drugs: Abscisic acid (B50724, purity>98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; γ-aminobutyric acid (L2002184, purity>99%) was purchased from Aladdin Company; Gallein (GC13945, purity>98%) was purchased from GLPBIO Company, USA; U73122 (T6243, purity=97.98%) was purchased from TargetMol Company, USA. Other items were the same as in Example 3.

[0171] 1.2.2 Main reagents: p-NF-κB p65 (AF2006) and NLRP3 antibody (DF7438) were purchased from Affinity Antibody Company; Cleaved caspase 1 (D210) antibody (40499) was purchased from Signalway Antibody Company, USA. Other reagents were the same as in Example 3.

[0172] 1.3 Methods

[0173] 1.3.1 Grouping and Drug Administration: MPC cells were routinely cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin, in a 5% CO2 incubator at 37 ℃. When growth was good, the medium was replaced with serum-free medium to synchronize the cell cycle for 12 h. Cells were then divided into a high glucose group (HG, 40 mM), a high glucose and quinine (50 µmol / L) co-treatment group, and groups treated with different inhibitors, including the TAS2R4 antagonists γ-aminobutyric acid (GABA, 12.5 µmol / L) and abscisic acid (ABA, 25 µmol / L), the Gβγ inhibitor Gallein (10 µmol / L), and the PLCβ2 inhibitor U73122 (10 µmol / L). Relevant parameters were measured after 48 h of cell culture in each group.

[0174] 1.3.2 Immunofluorescence assay for protein expression: Same as in Example 1.

[0175] 2 Results

[0176] 2.1 Effect of TAS2R4 antagonism on quinine-induced PLCβ2 protein expression in MPC cells under high glucose conditions

[0177] Literature reports that γ-aminobutyric acid (GABA) (Sai ​​P Pydi et al., J Biol Chem. 2014;289(36): 25054-66.) and abscisic acid (ABA) (Sai ​​P Pydi et al., Biochemistry. 2015;54(16):2622-31; Guanyin Yuan et al., Eur J Pharmacol. 2020;876:173063.) can antagonize the TAS2R4 receptor. In this study, we found that, compared with the HG+Qui group, co-treatment with 12.5 µmol / L GABA or 25 µmol / L ABA could reverse the effect of Qui increasing the expression of PLCβ2 protein in MPC cells induced by high glucose. The results are shown in […]. Figure 6 This indicates that both antagonists are effective at antagonizing the TAS2R4 receptor at the concentrations used.

[0178] 2.2 Effects of TAS2R4 antagonism on quinine-induced reduction of p-NF-κB p65 protein expression in MPC cells under high glucose conditions

[0179] We found that, compared with the HG group, Qui treatment significantly reduced the level of p-NF-κB p65 in the cytoplasm of MPC cells. Figure 7 In contrast to the HG+Qui group, co-treatment with the TAS2R4 antagonist GABA or ABA not only reversed the effect of Qui in reducing the p-NF-κB p65 level in the cytoplasm of MPC cells induced by high glucose, but also increased the p-NF-κB p65 level in the nucleus. (See results below.) Figure 7 These results indicate that TAS2R4 receptor inactivation mediates high glucose-induced increase in NF-κB p65 phosphorylation and nuclear translocation of NF-κB p65 in the cytoplasm of MPC cells.

[0180] 2.3 Effect of TAS2R4 signaling blockade on quinine-induced inhibition of NLRP3 inflammasome activation in MPC cells under high glucose conditions

[0181] Compared with the HG group, Qui treatment significantly reduced NLRP3 protein expression in MPC cells. Figure 8Compared to the HG+Qui group, co-treatment with the TAS2R4 antagonist ABA, the Gβγ inhibitor Gallein, or the PLCβ2 inhibitor U73122 eliminated the effect of Qui in reducing NLRP3 protein expression in MPC cells induced by high glucose. Figure 8 Furthermore, compared to the HG group, Qui treatment significantly reduced the expression of activated caspase 1 protein in MPC cells. Figure 9 In contrast to the HG+Qui group, co-treatment with the TAS2R4 antagonist ABA or the Gβγ inhibitor Gallein reversed the effect of Qui in reducing the expression of activated caspase 1 protein in MPC cells induced by high glucose. Figure 9 These results indicate that inhibition of the TAS2R4 / Gβγ pathway cancels the inhibitory effect of quinine on the NLRP3 inflammasome in MPC cells under high glucose conditions.

[0182] The above are merely preferred embodiments of the present invention and do not limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by achieving the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. The application of a TAS2R4 agonist in the preparation of a drug for the treatment and prevention of diabetic nephropathy; wherein the TAS2R4 agonist is quinine.

2. The application according to claim 1, characterized in that, This includes targeting the bitter taste receptor subtype 4, TAS2R4, as a drug target.

3. The application according to claim 2, characterized in that, The TAS2R4 agonist can activate the renal TAS2R4 receptor signaling.

4. The application according to claim 3, characterized in that, The TAS2R4 agonist can activate the TAS2R4 molecular signaling of glomerular podocytes to produce an anti-inflammatory effect and reduce damage to glomerular podocytes.

5. The application according to claim 3, characterized in that, The TAS2R4 agonist improved fasting blood glucose, body weight, and renal function in diabetic mice, and inhibited renal NLRP3 inflammasome and NF-κB. κ Activation of B.

6. The application according to claim 1, characterized in that, The TAS2R4 agonist can restore cell viability of mouse podocytes induced by high glucose, reverse the decrease in podocyte marker proteins nephrin and ZO-1, increase the protein expression of TAS2R4 and its downstream signaling molecule phospholipase Cβ2 (PLCβ2), and increase intracellular calcium ion levels.

7. The application according to claim 1, characterized in that, When mouse podocyte cell line MPC was co-treated with the TAS2R4 antagonist abscisic acid, the Gβγ inhibitor Gallein, and the PLCβ2 inhibitor U73122, the protein expression reduction effect of the TAS2R4 agonist on NLRP3 and Cleaved caspase 1 was significantly eliminated.

8. The application according to any one of claims 1-7, characterized in that, The TAS2R4 agonist is derived from natural products, chemically synthesized compounds, and microbial metabolites.

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